The broad objectives of this R21 RFA-DA-0 1-011 application are to extend the capabilities of the FLP-ERT and Cre-loxP site specific recombinase systems in mice by developing tools that will advance the following: 1) the ability to misexpress genes with greater temporal, spatial, and molecular precision, for example, in specific populations within populations of cells; 2) the ability to screen transgenics easily for recombinase activity, including FLP and Cre strains resulting from insertional mutagenesis; 3) the capacity to image aspects of cell morphology, while concomitantly relating this morphology to combinatorial patterns of gene expression; 4) the ability to determine cellular function (versus gene function) of specific cell populations. These capabilities are not presently available and would represent significant advancements. The studies described in this proposal concentrate on generating six widely useful recombinase target mouse strains that will extend the functionality of the many cell-specific FLP and Cre mice presently available. First, we will address the need for a high resolution dual responsive indicator mouse strain. While indicator strains exist that mark cells following either FLP- or Cre-mediated excision of disrupting DNA, no strains are presently available that achieve the higher resolution to specifically mark cells at the intersection of FLP and Cre expression domains (e.g. reflecting the overlap of two developmentally critical genes). Importantly, we will construct this dual responsive indicator strain using reporters designed to highlight cell morphology. From this dual responsive strain, we will go on to derive two broadly useful single, FLP or Cre, responsive indicator strains harboring properties not yet available. Second, a dual responsive ablation strain will be generated that allows for precise elimination of cell subpopulations in the embryonic and adult mouse. Development of a widely useful ablation strain will fill a significant technical gap by providing a powerful means to study the requirement for different cells in the development, structure, function, and aging of an organ system. From this strain we will go on to derive two broadly useful single, FLP or Cre, responsive ablation strains. Third, we will demonstrate the potential of the proposed six mouse strains by using them to extend our studies on the development and function of a brainstem system critical for movement control and sensorimotor transformations - the precerebellar afferent system. Because these novel mouse strains are designed to function in nearly all cell types, their impact on the generation of mouse models for human development and disease should be far-reaching.